extraction, purification and characterization of pyocyanin … · 2017-08-17 · international...

International Research Journal of

Vol. 6(5), 1-9, May (2017)

International Science Community Association

Extraction, purification and characterization of pyocyanin produced by

Pseudomonas aeruginosa Popy Devnath, Md. Kamal Uddin, Forkan Ahamed

Department of Microbiology, Faculty of Biological Sciences, University of Chittagong, Chittagong 4331, Bangladesh

Available online at: Received 13th March

Abstract

Pseudomonas aeruginosa, a gram negative bacteria, which exerts broad antagonistic activity against other

fungal pathogens through the production of a secondary metabolite

were taken for P. aeruginosa isolation. From sixteen primary P. aeruginosa

PP3) were selected, on the basis of pigmentation in cetrimide agar. The screening for antimicrobial activity of P

aeruginosain cross streak method showed that

Bacillus cereus (ATCC 14579) were sensitive, Escherichia coli (ATCC 8739) was intermediate, and K. pneumoniae (ATCC

43816) was resistant to the inhibitory action of the selected P. aeruginosa isolates. The antimicrobial pigment pyocyanin was

extracted from culture broth following solvent extraction method. Purification of the pigment was done by column and thin

layer chromatography. The Rf value was found around 0.81 for all the extracted pigment solution. Confirmation of the

pigment as pyocyanin was done through FTIR, and UV

functional groups (-OH, -C=N, -CH3 etc.) which belongs to the aromatic structure of pyocyanin. In UV

spectrophotometric analysis, a maximum absorption was observed at 270

enabled to increase pyocyanin production and the highest amount was produced by the isolate PU

concentration of 9.45 µg/mL. The antimicrobial activity of the purified pyocyanin

showed that highest concentration of pyocyanin (25

the lowest (5 µg/mL) was for S. aureus.

Keywords: Pyocyanin, Extraction, Purification, Antimicrobial activity,

Introduction

Pseudomonas aeruginosa is a Gram negative aerobic rod and an

opportunistic pathogen. It is widespread in the terrestrial and

aquatic environment and can cause infection in

organisms1. Chronic infections in cystic fibrosis patients and

wound infections especially of burns are also associated with

this pathogen2. It is now obvious that some phenazine like

bioactive compounds have antimicrobial activity and

aeruginosa frequently produce such compounds. Among the

recognized phenazine pigments, pyocyanin, 1

phenazine, phenazine-1-carboxamide and phenazine

carboxylic acid are notable3. It has been found that phenazine

compounds are associated with biofilm development, such as

Phenazine-1-Carboxylic acid, which is secreted by some

pseudomonads, promote bacterial biofilm formation via

acquisition of ferrous iron4. Pyocyanin also has significance as

quorum sensing (QS) signaling molecule as well as a virul

factor for P. aeruginosa. Although pseudomonads are

frequently reported for their pathogenicity, the ability of this

microorganism to produce antimicrobial pigment opened the

door of using this as biological control agent. Researchers

invented an appreciable number of new antimicrobial agents in

the last three decades, bacterial resistance to antimicrobial

Journal of Biological Sciences ___________________________

Association


Pseudomonas aeruginosa and evaluation for its antimicrobial activityPopy Devnath, Md. Kamal Uddin, Forkan Ahamed, Md. Towhid Hossain and Mohammed Abul Manchur


[email protected]

Available online at: www.isca.in, www.isca.me March 2017, revised 5th April 2017, accepted 2nd May 2017

Pseudomonas aeruginosa, a gram negative bacteria, which exerts broad antagonistic activity against other

fungal pathogens through the production of a secondary metabolite-pyocyanin. In the present study, various clinical samples

From sixteen primary P. aeruginosa isolates, five isolates (PS

) were selected, on the basis of pigmentation in cetrimide agar. The screening for antimicrobial activity of P

aeruginosain cross streak method showed that Staphylococcus aureus (ATCC 6538), Salmonella enterica (NCTC 6017), and

CC 14579) were sensitive, Escherichia coli (ATCC 8739) was intermediate, and K. pneumoniae (ATCC


owing solvent extraction method. Purification of the pigment was done by column and thin

value was found around 0.81 for all the extracted pigment solution. Confirmation of the

pigment as pyocyanin was done through FTIR, and UV-visible spectrophotometric analysis. FTIR analysis revealed

CH3 etc.) which belongs to the aromatic structure of pyocyanin. In UV

spectrophotometric analysis, a maximum absorption was observed at 270-275nm.The modification of media composition

enabled to increase pyocyanin production and the highest amount was produced by the isolate PU

The antimicrobial activity of the purified pyocyanin pigment against

showed that highest concentration of pyocyanin (25 µg/mL) was needed to produce zone of inhibition in case of E. coli, and

Pyocyanin, Extraction, Purification, Antimicrobial activity, Characterization.

is a Gram negative aerobic rod and an

opportunistic pathogen. It is widespread in the terrestrial and

infection in a wide range of

. Chronic infections in cystic fibrosis patients and

wound infections especially of burns are also associated with

. It is now obvious that some phenazine like

bioactive compounds have antimicrobial activity and P.

frequently produce such compounds. Among the

recognized phenazine pigments, pyocyanin, 1-hydroxy

carboxamide and phenazine-1-

. It has been found that phenazine

ilm development, such as

Carboxylic acid, which is secreted by some

promote bacterial biofilm formation via

Pyocyanin also has significance as

quorum sensing (QS) signaling molecule as well as a virulence

. Although pseudomonads are

frequently reported for their pathogenicity, the ability of this

microorganism to produce antimicrobial pigment opened the

door of using this as biological control agent. Researchers

eciable number of new antimicrobial agents in

the last three decades, bacterial resistance to antimicrobial

agents has also increased simultaneously. Common

antimicrobial agents are not working properly against those

infectious organisms5. The present stud

some high pyocyanin yielding P. aeruginosa

antimicrobial pigment from its culture, purify the pigment,

augment its production and test its antimicrobial activity.

Materials and methods

Isolation and identification of P. aeruginosasamples: To isolate P. aeruginosa

samples (burned skin swab, urine and pus) Pseudomonas

Cetrimide Agar (OxoidTM

) was used as selective media

first, samples were enriched in Brain

medium (OxoidTM

). Then the enriched samples were cultured on

cetrimide agar by streak plate and pour plate method, observed

for distinguishing pigmentation and 5 isolates (

PU10 and PP3) were selected from 16 initial isolates on

of visual vigorous pigmentation for further study. By comparing

the microscopic features, physiological and biochemical

characteristics of the isolates with the standard description given

in “Bergey's Manual of Determinative Bacteriology”, these

were identified as P. aeruginosa7.

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Int. Res. J. Biological Sci.

1


and evaluation for its antimicrobial activity , Md. Towhid Hossain and Mohammed Abul Manchur

*


Pseudomonas aeruginosa, a gram negative bacteria, which exerts broad antagonistic activity against other bacterial and

pyocyanin. In the present study, various clinical samples

isolates (PS1, PU5, PU8, PU10 and

) were selected, on the basis of pigmentation in cetrimide agar. The screening for antimicrobial activity of P

Staphylococcus aureus (ATCC 6538), Salmonella enterica (NCTC 6017), and

CC 14579) were sensitive, Escherichia coli (ATCC 8739) was intermediate, and K. pneumoniae (ATCC


owing solvent extraction method. Purification of the pigment was done by column and thin

value was found around 0.81 for all the extracted pigment solution. Confirmation of the

visible spectrophotometric analysis. FTIR analysis revealed different

CH3 etc.) which belongs to the aromatic structure of pyocyanin. In UV-Vis

modification of media composition

enabled to increase pyocyanin production and the highest amount was produced by the isolate PU10 in Medium-B having a

pigment against different test organism

g/mL) was needed to produce zone of inhibition in case of E. coli, and

agents has also increased simultaneously. Common

antimicrobial agents are not working properly against those

. The present study was designed to isolate

P. aeruginosa isolates to extract

antimicrobial pigment from its culture, purify the pigment,

augment its production and test its antimicrobial activity.

P. aeruginosa from clinical P. aeruginosa from various clinical

samples (burned skin swab, urine and pus) Pseudomonas

) was used as selective media6. At

first, samples were enriched in Brain-Heart infusion (BHI) broth

). Then the enriched samples were cultured on

cetrimide agar by streak plate and pour plate method, observed

for distinguishing pigmentation and 5 isolates (PS1, PU5, PU8,

) were selected from 16 initial isolates on the basis

of visual vigorous pigmentation for further study. By comparing

the microscopic features, physiological and biochemical

characteristics of the isolates with the standard description given

in “Bergey's Manual of Determinative Bacteriology”, these

International Research Journal of Biological Sciences ________________________________________________ISSN 2278-3202

Vol. 6(5), 1-9, May (2017) Int. Res. J. Biological Sci.

International Science Community Association 2

Primary screening for the isolates having antimicrobial

activity: Cross streak method was primarily followed to

investigate the antimicrobial activity of the selected isolates8.

Here, the following organisms - S. aureus (ATCC 6538), B.

cereus (ATCC 14579), E. coli (ATCC 8739), S. enterica

(NCTC 6017), K. pneumoniae (ATCC 43816) and P.

aeruginosa (ATCC 9027) - were used as test organism. A clear

zone of growth inhibition near the adjoining culture line of P.

aeruginosa and test organisms would be indicative of

pseumonads’ antimicrobial activity.

Extraction, purification and characterization of the pigment

produced by Pseudomonas isolates: The selected

Pseudomonas isolates were grown first in the Pseudomonas

broth at 37oC for 48 hours for pigment production. Pigment rich

broth culture was then centrifuged at 10000 rpm for 15 min and

the supernatant was collected. It was then filtered through 0.45

µm pore sized membrane filter and used as crude extract.

Extraction of pigment from crude extract was done using

chloroform and HCl as Ingledew and Campbell, 1969 with some

modification9. Chloroform was added in the culture broth at the

ratio of (2:1) and after vortexing a blue solvent layer was

produced. The blue layer was collected, then 0.1N HCl solution

(20% of the blue layer’s volume), was added and vortexed,

which produced an acidified pink upper layer. The pink layer

was then neutralized with Tris-Base and the neutralized layer

was again treated with chloroform. The whole procedure was

repeated several times to make it pure.

Purification of the extracted pigment was done by column

chromatography (column size 45 x 3.5 cm) using silica gel G, as

stationary phase and a solution of methanol and chloroform at

the ratio of 1:1 as mobile phase10

. The eluted sample was further

analyzed by TLC using methanol and chloroform at the ratio of

1:1 as mobile phase to check its purity and the Rf value of the

partially purified pigment was checked. The fraction of the

eluted sample was also treated with 0.1 N NaOH and kept for 2

-3 hours for crystallization11

.

The crystals were separated by membrane filtration, dried and

observed under microscope. The crystals was stored and

suspended in water when necessary. Extracted pigment solution

from the 5 efficient isolates of P. aeruginosa were subjected to

UV-visible spectrophotometer12,13

and a maximum absorbance

was recorded by UV-1800 UV-VIS Spectrophotometer

(Shimadzu). Fourier Transform Infrared (FTIR) spectroscopic

analysis of the pigment was also done using potassium bromide

(KBr) as a window material14

.

Augmentation of pigment production: Pigment production

was primarily done by using Pseudomonas broth. Then it was

augmented using a modified Pseudomonas broth (Glycerol 1ml,

MgCl2 0.14g, K2SO4 1g, Asparagine 0.1%, Peptone 2g, Distilled

water upto100ml) which we named Medium-A and Glycerol-

alanine broth (Glycerol 1ml, MgCl2 0. 14g, K2SO4 1g, D-alanine

0.1%, Peptone 2g, distilled water 100ml) which we named

Medium-B. 48 hours of incubation period was needed for

vigorous pigmentation. After pigmentation, following the

extraction procedure, the concentration (µg/mL) of the pigment

in extracted solution was determined by measuring the optical

density (absorbance) at 520 nm wavelength using a UV-visible

spectrophotometer (Shimadzu) and multiplying the optical

density value (OD520) with 17.07215,16

.

Determination of antimicrobial activity of the purified

pigment: The antibacterial activity of the purified pigment

produced by the selected isolates against different bacterial

strains - S. aureus (ATCC 6538), S. enterica (NCTC 6017), E.

coli (ATCC 8739) and B. cereus (ATCC 14579) - was

investigated following agar well diffusion method17

. Mueller-

Hinton Agar was used as agar medium and different

concentration of the purified pigment – 5, 10, 15, 20, 25, 30, 35,

40 and 45 µg/ml in water was prepared to test the antibacterial

activity. First the prepared media was seeded with the test

organism (OD equivalent to 0.5 McFarland), poured in the

petriplate, solidified and wells were made in the agar medium.

Then 100 µL of each prepared pigment solutions were poured in

different wells of the agar plate and incubated for 24 hours at

37ºC. After incubation the diameter of zone of growth inhibition

was measured to determine the antimicrobial activity of the test

agent. Each experiment was replicated trice.

Results and discussion

Isolation and identification of P. aeruginosa from clinical

samples: Three types of colony morphology was observed on

the selective medium Pseudomonas Cetrimide Agar–colonies

with vigorous yellow-green pigmentation (31%), colonies with

scanty pigmentation (38%) and no pigmentation (31%). The

selective media contain a quaternary ammonium compound,

cetrimide, which has broad spectrum bactericidal activity

against a wide range of Gram-positive and some Gram-negative

microorganisms. A number of water soluble iron chelators, like

the yellow-green or yellow-brown fluorescent pyoverdin are

also produced by P. aeruginosa.

The characteristic bright green colour of P. aeruginosa broth

culture is developed when water-soluble blue pyocyanin

combines with pyoverdin6. We have found 5 isolates having

such pigmentation characteristics and initially recognized as P.

aeruginosa. Among these isolates 3 were from urine and

remaining each isolate from burned skin swab and pus. P.

aeruginosa is an opportunistic pathogen and responsible for

nosocomial infection of urinary tract and burn patients18

.

Their identification was confirmed by microscopic observation

and biochemical tests as noted in Table-1 and it was also found

that all the isolates had the same characteristics. The organisms

have diverse metabolic and physiological ability like catalase

activity, sugar fermentation, growing at different temperature

and pH.




Table-1: Morphological, cultural, biochemical and physiological characteristics of the selected isolates.

Characteristics Isolates

PS1 PU10 PU8 PU5 PP3

Gram staining negative negative negative negative negative

Oxidase test positive positive positive positive positive

Catalase test positive positive positive positive positive

Motility test motile motile motile motile motile

Citrate utilization positive positive positive positive positive

Gelatin hydrolysis positive positive positive positive positive

Deep glucose agar test positive positive positive positive positive

Casein hydrolysate test positive positive positive positive positive

Voges-Proskauer test negative negative negative negative negative

Methyl red test negative negative negative negative negative

Nitrate reduction test negative negative negative negative negative

H2S production test negative negative negative negative negative

Indole test negative negative negative negative negative

Glucose fermentation Acid Acid Acid Acid Acid

Fructose fermentation Acid Acid Acid Acid Acid

Mannitol fermentation Acid Acid Acid Acid Acid

Arabinose fermentation No acid, no gas No acid, no gas No acid, no gas No acid, no gas No acid, no

gas

Sucrose fermentation No acid, no gas No acid, no gas No acid, no gas No acid, no gas No acid, no

gas

Growth response at different pH

pH-5 + + + + +

pH-7 ++ ++ ++ ++ ++

pH-8 +++ +++ +++ +++ +++

pH-9 +++ +++ +++ +++ +++

Growth response at different temperature (ºC)

4 ºC - - - - -

30 ºC +++ +++ +++ +++ +++

37 ºC +++ +++ +++ +++ +++

42 ºC ++ ++ ++ ++ ++

Note: - = no growth, + = scanty growth, ++ = moderate growth, and +++ = heavy growth.

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Vol. 6(5), 1-9, May (2017)



activity: P. aeruginosa synthesizes various bioactive substances

among these pyocyanin is the best known for its antimicrobial

activity against S. aureus, S. epidermis, Clostridium botulinum,

Micrococcus luteus, B. subtilis, as well as B. licheniformis20

. In the present study of primary screening for antimicrobial

activity of P. aeruginosa against different test organism

following cross streak method we found,

6538), B. cereus (ATCC 14579), and Salmonella enterica

(NCTC 6017) showed visually maximum sensitiv

in Figure-1. E. coli (ATCC 8739) showed intermediate

sensitivity, K. pneumonia (ATCC 43816) was weakly sensitive

and P. aeruginosa (ATCC 9027) was not sensitive at all. In a

previous study, it was found that P. aeruginosa

antagonistic activity against S. aureus, E. coli, P. vulgaris,

Bacillus sp. in cross streak method13

. Machan also found anti

staphylococcal activity of P. aeruginosa with cross streak test.

Prevention of Gram negative bacterial growth

was also recorded21

. For example, E. coli Enterobacter cloacae

Serratia marcescens, Salmonella typhi, Vibrio

parahaemolyticus, Proteus mirabilis, Proteus

Paracoccus denitrificans, Citrobacter sp.

carotovora22,23

. This metabolite exhibits not only antibacterial

effect but also anti-parasitic activity against the nematode

Caenorhabditis elegans24,25

.


produced by Pseudomonas isolates:

pseudomonas broth turned into bluish green or yellowish green

after vigorous pigmentation within the given incubation period.

Pseudomonas aeruginosa produce different pigments, including

pyocyanin (blue green), pyomelanin (light-brown), pyoverdin

(yellow, green and fluorescent) and pyorubrin (red

which are responsible for this colour change elaborated by

pseudomonads27

. Following the extraction procedure we found a

blue colour solution in chloroform that turned into pink

upon addition of 0.1 N HCl. Pyocyanin is soluble in chloroform

producing blue colour10

. It is considered as a resonance hybrid

of the mesomeric forms of N-methyl-1-hydroxyphenazine and is

capable of undergoing a two-electron reduction to a colourless

product, leukopyocyanin. It can exist either in

oxidized form. The reduced form of pyocyanin was unstable

rapidly reacts with molecular oxygen28

. The pigment is wine

at acid condition because of the basic property of one of the

nitrogen atoms and blue at alkaline reaction29

.

In the column chromatographic analysis we found only a single

blue colour band during separation indicating the presence of no

other pigment in the extracted solution. The TLC

extract also substantiates the findings in column

chromatography. A blue spot with Rf value ranging from 0.70 to

0.81 was observed for the pigment extracted from all the

isolates which was characterized as phenazine compound such

as pyocyanin13

. Following crystallization process, t

produced crystal which was needle like when observed under

microscope (Figure-2). In 2011, El-Shouny reported that 0.1M

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synthesizes various bioactive substances

among these pyocyanin is the best known for its antimicrobial

S. epidermis, Clostridium botulinum,

B. licheniformis11,19,

udy of primary screening for antimicrobial

against different test organism

following cross streak method we found, S. aureus (ATCC

Salmonella enterica

(NCTC 6017) showed visually maximum sensitivity as shown

(ATCC 8739) showed intermediate

(ATCC 43816) was weakly sensitive

(ATCC 9027) was not sensitive at all. In a

P. aeruginosa showed

S. aureus, E. coli, P. vulgaris,

. Machan also found anti-

with cross streak test.

Prevention of Gram negative bacterial growth by this molecule

E. coli Enterobacter cloacae,

Salmonella typhi, Vibrio

Proteus vulgaris,

sp. and Erwinia

s metabolite exhibits not only antibacterial

parasitic activity against the nematode


isolates: The inoculated

s broth turned into bluish green or yellowish green

after vigorous pigmentation within the given incubation period.

produce different pigments, including

brown), pyoverdin

(yellow, green and fluorescent) and pyorubrin (red-brown)26

which are responsible for this colour change elaborated by

. Following the extraction procedure we found a

blue colour solution in chloroform that turned into pink-red

of 0.1 N HCl. Pyocyanin is soluble in chloroform

It is considered as a resonance hybrid

hydroxyphenazine and is

electron reduction to a colourless

It can exist either in reduced or

oxidized form. The reduced form of pyocyanin was unstable and

. The pigment is wine-red

at acid condition because of the basic property of one of the

.

In the column chromatographic analysis we found only a single

blue colour band during separation indicating the presence of no

other pigment in the extracted solution. The TLC analysis of the

the findings in column

value ranging from 0.70 to

0.81 was observed for the pigment extracted from all the

isolates which was characterized as phenazine compound such

. Following crystallization process, the pigment

needle like when observed under

Shouny reported that 0.1M

NaOH treatment for 2 hours in the chloroform extract of

pyocyanin forms needle like crystals

Figure-1: Antimicrobial activity of

(1) S. enterica NCTC 6017 (2) S. aureus

cereus ATCC 14579.

Figure-2: Microscopic observation of needle like crystals of the

pigment pyocyanin formed in the crystallization process (1

The UV-visible spectrophotometric analysis of extracted

purified pigment dissolved in 0.1 N HCl showed that peak was

observed at a maximum range of 270

results are almost congruent with those of

found that UV-visible spectra of pyocyanin was around 278

nm13,14

.

The FTIR spectra, as in Figure-4, of the extracted compound in

different selected isolates shows bands between 3400

which indicate the presence of –OH group and the appearance

of bands between 3000-2900 is an indication of C

aromatic compound30

. As an Aromatic hydrocarbon the

compound shows absorptions in the regions 15001and1600-1585 cm

-1 due to vibrations of carbon

stretching in the aromatic ring. Absorption between 1590

cm-1

and 1280- 1250 cm-1

are indicative of C=N bonds and C

bonds respectively in aromatic stretching. In case of PP

a hump appeared at 1630 cm-1

which c

bond. The presence of –CH3 group is confirmed with the

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NaOH treatment for 2 hours in the chloroform extract of

pyocyanin forms needle like crystals11

.

Antimicrobial activity of P. aeruginosa (PU5) against

S. aureus ATCC 6538 (3) B.

Microscopic observation of needle like crystals of the

pigment pyocyanin formed in the crystallization process (10X).

visible spectrophotometric analysis of extracted

0.1 N HCl showed that peak was

observed at a maximum range of 270-275 nm (Figure-3). These

results are almost congruent with those of Ohfuji et al. who

visible spectra of pyocyanin was around 278

4, of the extracted compound in

different selected isolates shows bands between 3400-3300 cm-1

OH group and the appearance

2900 is an indication of C-H stretch for

. As an Aromatic hydrocarbon the

compound shows absorptions in the regions 1500-1400 cm-

to vibrations of carbon- carbon

stretching in the aromatic ring. Absorption between 1590-1600

are indicative of C=N bonds and C-N

bonds respectively in aromatic stretching. In case of PP3 sample

which can be assigned for C=N

group is confirmed with the –C-H

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Vol. 6(5), 1-9, May (2017)


stretches of the alkyl (methyl) group in the 1380

range. It shows no peak at 1690-1760 cm-

compound has no chance to have a C=O group in its struc

Comparing the spectra, presented at Table-2, it was averred that

the purified pigment was pyocyanin since most of the functional

groups present in pyocyanin structure were found here.

Figure-3: UV absorption spectra of the extracted

dissolved in 0.1N HCl.

Figure-4: FTIR spectra of the pyocyanin pigment extracted

from the isolate P. aeruginosa PU8.

Augmentation of pigment production: Carbon and nitrogen

sources highly affect the production of pyocyanin. Production of

pyocyanin was once carried out in King’s Medium

later modified by combining different amino acids, like

asparagine, alanine, tyrosine and leucine, in the culture medium

which resulted in a more vigorous pigment production based on

King’s Medium26,31

. At present study, Pseudomonas broth was

modified by adding 0.1% asparagine (Medium

production. The addition of both glyceroland alanine as

substrates in Medium-B augmented more pyocyanin

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stretches of the alkyl (methyl) group in the 1380-1400 cm-1

-1 that means the

compound has no chance to have a C=O group in its structure.

2, it was averred that

the purified pigment was pyocyanin since most of the functional

groups present in pyocyanin structure were found here.

the extracted pigment

FTIR spectra of the pyocyanin pigment extracted

Carbon and nitrogen

sources highly affect the production of pyocyanin. Production of

carried out in King’s Medium27

. It was

later modified by combining different amino acids, like

tyrosine and leucine, in the culture medium

which resulted in a more vigorous pigment production based on

. At present study, Pseudomonas broth was

modified by adding 0.1% asparagine (Medium-A) for pyocyanin

production. The addition of both glyceroland alanine as

B augmented more pyocyanin production

than Medium-A with asparagine. Inorganic ion such as K

Mg2+

, PO43-

, SO42-

, Fe2+

were essential for pyocyanin

production. So in the Medium-B, K

ions were included and it was

pigmentation than Medium A. Both medium contained MgCl

which also increase pigment formation

the isolates had better pigmentation in Medium

A. The isolate PU10 produced highest amount of py

(9.45 µg/mL) in Medium-B whereas the lowest (2.79

produced by PS1 in Medium-A as shown in Figure

and nitrogen sources in the growth media affect the production

of pyocyanin but most nutrients support pyocyanin

as long as the phosphate ion concentration is low; high

phosphate concentration had been shown to inhibit pyocyanin

production33

. The presence of Sulfate (SO

pyocyanin synthesis, while sulfite (SO

the presence of Ca2+

ion increases the synthesis from three to

five folds. Production of pyocyanin was encouraged by the

addition of 1% (weight/volume) of sodium citrate

of this pigment also appears to be under the control of iron

concentration since addition of iron to a medium containing low

phosphate stimulates the synthesis of pyocyanin and related

phenazine pigments by other species of bacteria.

alanine, leucine, amino acids stimulate the production of

pyocyanine31

. It is also noticeable that all the

isolates (PU5, PU8 and PU10) isolated from urine sample

produced more pyocyanin than those isolates like PS1and PP3

isolated from burned skin swab and pus respectively. There

might be some relationship among the pseudomonads

environment and their pyocyanin productivity.

Table-2: FTIR spectroscopic analysis of the pigment extracted

from different isolates.

Functional

groups

FTIR Spectra of pigment extracted from

PU10 PS1

-OH 3429.43 3429.43

-C=N 1620.21 1627.92

-C=C 1604.7 1562.34

-C-H 2924.09 2924.09

C-O 1319.31 1354.03

-C-N 1265.30 1261.45

Methyl

groups 1384.89 1384.89

Note: h = hump

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5

A with asparagine. Inorganic ion such as K+,

were essential for pyocyanin

B, K+

, Mg2+

, PO43-

, SO42-

, Fe2+

seen that it gives better

pigmentation than Medium A. Both medium contained MgCl2

which also increase pigment formation32

. It was found that all

the isolates had better pigmentation in Medium-B than Medium-

produced highest amount of pyocyanin

B whereas the lowest (2.79 µg/mL) is

A as shown in Figure-5. Carbon

and nitrogen sources in the growth media affect the production

of pyocyanin but most nutrients support pyocyanin production

as long as the phosphate ion concentration is low; high

phosphate concentration had been shown to inhibit pyocyanin

The presence of Sulfate (SO4) increased the

pyocyanin synthesis, while sulfite (SO3) inhibited its synthesis;

ion increases the synthesis from three to

five folds. Production of pyocyanin was encouraged by the

addition of 1% (weight/volume) of sodium citrate34

. Synthesis

of this pigment also appears to be under the control of iron

nce addition of iron to a medium containing low

phosphate stimulates the synthesis of pyocyanin and related

phenazine pigments by other species of bacteria. Tyrosine,

alanine, leucine, amino acids stimulate the production of

able that all the P. aeruginosa

) isolated from urine sample

produced more pyocyanin than those isolates like PS1and PP3

isolated from burned skin swab and pus respectively. There

might be some relationship among the pseudomonads’ hosting

environment and their pyocyanin productivity.

FTIR spectroscopic analysis of the pigment extracted

FTIR Spectra of pigment extracted from

PU8 PP3 PU5

3348.42 3348.42 3348.42

1627.92 1630h 1627.92

1589.34 1589.34 1550.77

2920.23 2920.23 2943.37

1342.46 1342.46 1342.46

1288.45 1288.45 1261.45

1396.46 1400.32 1396.46




Figure-5: Rate of pyocyanin production by the selected isolates

(PS1, PU5, PU8, PU10 and PP3) in Medium-A and Medium B.

Figure-6: Effect of different concentration (µg/mL) of

pyocyanin on the growth of S. aureus ATCC 6538.

Figure-7: The antimicrobial activity of different concentration (µg/mL) of pyocyanin against different test microorganisms. Here,

S. aureus was most sensitive to pyocyanin and produced zone of inhibition at lowest concentration (5 µg/mL) of pyocyanin and E.

coli required highest concentration (25 µg/mL) of pyocyanin.




Antimicrobial activity of the purified pyocyanin: Previously

in the primary screening process, we have found that P.

aeruginosa have good antimicrobial activity against gram

positive and gram negative bacteria. The subsequent

antimicrobial activities of purified pigment corroborate the

initial findings in the primary screening. Here we found that the

bactericidal effect of pyocyanin that we observed was

concentration dependent in all cases. The Figure-6 and 7

showed that among the test organisms S. aureus was the most

sensitive to pyocyanin and it was inhibited at 5 µg/mL of

pyocyanin producing 17.5 mm of growth inhibition zone. B.

cereus, Salmonella enteric and E. coli showed zone of growth

inhibition at 10, 15 and 25 µg/mL of pyocyanin respectively.

From the results it is clear that gram negative bacteria were less

sensitive than gram positive bacteria. It was reported earlier that

purified pyocyanin showed good antimicrobial property against

S. aureus, E. coli, Klebsiella sp, S. typhi, Shigella sp. C.

albicans14

. The lowest MIC of purified pyocyanin was reported

as 20 µg/ml whereas the highest MIC was 50 µg/ml was against

E. coli12

. In 1981, Baron and Rowe proposed that around 2.9 µg

of purified pyocyanin is sufficient for inhibiting bacterial

growth where gram positive Micrococcus luteus, S. aureus and

Bacillus licheniformnis showed maximum zone of inhibition

and P. aeruginosa remained resistant to pyocyanin action10

.

The present study depicts the antimicrobial activity exerted by

purified pyocyanin which is dependent on concentration. It was

revealed in earlier reports that pyocyanin inhibits bacterial

growth by interrupting active metabolic transport in bacterial

cells through the interaction with respiratory chain10

. Pyocyanin

upon up-taking an electron, produce a relatively stable anion

radical and readily undergo a redox cycle which is responsible

for its antagonistic action. Pyocyanin itself becomes reduced

and reduces oxygen univalently to toxic superoxide radical

which are actually responsible for its antimicrobial activity35

.

The bacteria which are resistant to pyocyanin have the ability to

produce catalase and superoxide dismutase and the resistance is

also contingent on presence of oxygen. It is surprising that

although P. aeruginosais a strictaerobe, it is resistant to

pyocyanin and readily evade the injury caused by toxic free-

radical produced during pyocyanin synthesis by itself or

exposed in the environment28

.

Pyocyanin has enormous antimicrobial potentiality to be used as

antibacterial, antifungal, antiprotozoal agent as well as nitric

oxide antagonist. The inhibitory action of pyocyanin against

different fungi like Aspergillussp, Candida albicans, some

pathogenic yeast and phytopathogens was reported in earlier36

.

The antiprotozoal activities of pyocyanin was studied by Dive

found that pyocyanin inhibited the growth and division of

Colpidiumcampylum37

. In various pharmacological preparations,

it is used as a nitric oxide (NO) antagonist and has various

pharmacological effects38

. It has also been reported that it has

anticancer activity on growth of cancer cells such as Leukemic

cells that was inhibited when treated with pyocyanin, more over

induce apoptosis on neutrophils39

.

Conclusion

In this study, it has been found that pyocyanin pigment is a good

antimicrobial agent, as it can inhibit both gram positive and

gram negative bacteria, such as S. aureus, B. cereus E.coli and

S. enterica. Antimicrobial resistance is an alarming issue to deal

with, as most of the pathogen becoming resistant to their

common treatable antibiotic. Recent studies report that S. aureus

and E. coli are multidrug resistant bacteria. Inhibition of these

and others like pathogens by pyocyanin, expresses its

importance and potentiality as an antimicrobial agent. It can be

used as therapeutic agent to treat infections caused by these

pathogenic bacteria.

Acknowledgements

The authors acknowledged their gratitude to Research and

Development Office, University of Chittagong, Bangladesh for

funding in this research work under the revenue budget of the

year 2014-2015. We also deeply grateful to Dr. Tapashi Ghosh

Roy, Professor, Department of Chemistry, University of

Chittagong for providing FTIR spectroscopy facility.

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